Distantly metastatic dedifferentiated thyroid cancer is a fatal disease without effective treatment. This is from loss of ability to concentrate iodide, rendering it unresponsive to radioactive iodine, the only known systemic therapy. Iodide uptake requires expression of sodium-iodide symporter (hNIS). Hypermethylation of hNIS promoter region was thought to be a likely mechanism for loss of hNIS expression in thyroid cancer cells. hNIS expression and function is restored after treating these cells with demethylating and histone deacetylase-inhibiting agents. It was presumed that these agents permit transcriptional machinery access to the gene by relaxing chromatin compaction. 5-azacytidine (AzaC;DNA methyltransferase inhibitor) and sodium butyrate (NaB;putative histone deacetylase inhibitor) both, singly and together, restore hNIS expression and function to thyroid cancer cell lines. To show relaxed hNIS chromatin structures suggested by their presumed epigenetic effects, we assessed DNAse I digestion rate of genomic hNIS regions when AzaC and NaB restored hNIS mRNA expression and hNIS function. Surprisingly, these treatments did not produce enhanced DNAse I sensitivity suggesting the absence of effect on chromatin compaction. NaB-treated dedifferentiated thyroid cancer cells, transfected with a luciferase reporter construct containing hNIS gene promoter, stimulated both luciferase expression and native hNIS expression. Treating these cells with protein synthesis inhibitors (PSIs;cycloheximide, anisomycin, emetine), stimulated luciferase expression in a dose-dependent, time course-dependent, cell type-specific, and promoter-specific fashion;as well as restored endogenous hNIS gene mRNA expression. This suggested presence of a trans-acting transcriptional inhibitor, NIS-repressor, responsible for loss of hNIS expression. We mapped the hNIS promoter sequence region responsible for this effect, the NIS-repressor binding site (NRBS). NRBS, used as a radiolabeled probe in electrophoretic mobility shift assays with thyroid cancer cell nuclear extract, shows sequence-specific protein binding. These proteins were analyzed with liquid chromatography coupled to tandem mass spectrometry and revealed identity of a significant component of NIS-repressor. We hypothesize that loss of iodide transport activity in thyroid cancer results from loss of hNIS gene expression, consequent to NIS-repressor binding to the hNIS promoter. This novel mechanism may underlie or be in addition to presumed epigenetic mechanisms of hNIS gene reactivation reported with various pharmacologic agents. Treatments directed to reduce or antagonize NIS- repressor components should restore hNIS gene expression responsible for iodide transport enabling I-131 therapy of dedifferentiated thyroid cancers. Our aims are to confirm our identification of NIS-repressor protein and identify other protein complex components, discern its physiological and pathophysiological effects, and develop strategies targeting it. This should result in treatments to restore NIS activity in dedifferentiated thyroid cancers, restoring use of radioactive iodine to treat thyroid cancer metastases. Public Health Relevance Statement: Thyroid cancer, for unknown reasons, is the most rapidly increasing cancer in the United States in both men and women and it is the most common cancer in white women between 20 and 30 years of age. Although 75% of thyroid cancer patients respond adequately to radioactive iodine therapy, 10% of patients lose response to this treatment and eventually die from disseminated tumor. Development of new methods of restoring clinical response to radioactive iodine could save lives.